Hematopoietic stem cells (HSC), largely residing in the bone marrow of adult mammals, are the progenitors of all blood cells, including all myeloid and lymphoid lineages. Unlike many other adult stem cells, the presence of an HSC within a population of cells can be assessed definitively. This is accomplished by transplantation experiments, where donor cells are injected intravenously into lethally irradiated, immunodeficient recipient mice. A single HSC can reconstitute the complete hematopoietic system for up six months. Indeed, HSCs are responsible for the repopulation of the hematopoietic compartment of bone marrow transplantation patients. While HSC transplantation remains the most clinically successful stem cell treatment, the availability of HSCs suitable for therapy is limited. Pluripotent stem cells (PSC), functionally defined by their ability to differentiate into all cells of an organism and their unlimited self-renewal, are an appealing starting point for the in vitro derivation of HSCs. PSC-derived HSCs (PSC-HSC) would be invaluable as a platform to model the genetic programs that govern the HSC and production of the hematopoietic compartment, as well as hematological diseases. Furthermore, with the advent of induced pluripotent stem cells, patient-specific PSC- HSC ultimately may be used to treat a variety of diseases currently treated (when possible) with allogeneic or autologous bone marrow transplants, especially hematological malignancies. The Daley lab first produced transplantable mouse PSC-HSCs by directed differentiation of ESCs, ectopic expression of the homeobox transcription factor HoxB4, and co-culture with OP9 stromal cells (Kyba et al 2002). While these PSC-HSCs make multilineage contributions to the host hematopoietic compartment, the lymphoid potential and long-term engraftment of these cells were limited. Reasoning that a better understanding of the developmental programs that orchestrate and specify the HSC in vivo would guide a more faithful derivation of PSC-HSCs, we performed gene expression profiling of purified HSCs and their immediate precursors at key developmental stages from the mouse embryo (McKinney-Freeman et al 2012). From this study, we learned that the Hoxb4- induced PSC-HSCs were globally more similar to the definitive HSCs of the adult than the more primitive HSCs of the early embryo, suggesting that only a limited set of programs distinguish PSC-HSC from HSC. The studies proposed here will determine the contribution of two candidate genetic programs to the limited self- renewal and lymphoid potential of current PSC-HSC. Aim 1 will determine the functional consequences of posterior HoxA expression in Hoxb4-induced PSC-HSCs. Aim 2 will assess the contribution of Notch signaling to deficient lymphopoiesis in Hoxb4-induced PSC-HSCs. The five-year training program has been designed to establish Dr. Patrick Cahan as an independent investigator in Stem Cell Biology. The proposed research and training program will be carried out in Dr. George Daley's lab at Boston Children's Hospital in the Division of Hematology/Oncology, which is also home to the laboratories of Drs. Orkin, Williams, and Zon, internationally known investigators who have consistently produced ground-breaking research in the areas of stem cell biology, development, hematopoiesis, chemical biology, transcription, cell cycle, and cancer genetics. The candidate was trained as a Computational Biologist in graduate school at Washington University in St. Louis, where he investigated the functional consequences of DNA copy number variation in hematopoietic stem and progenitors. As a postdoctoral fellow in the Daley Lab at Boston Children's Hospital, Dr. Cahan's central area of research is elucidating the differences between in vivo blood development and directed differentiation of blood from PSCs with the long-term goal of improving the fidelity with which in vitro derived populations mimic their in vivo counterparts. To become an interdisciplinary and independent investigator, and to complete the proposed research, the candidate needs to solidify his foundation of Developmental Biology, to expand his experimental expertise (e.g. FACS analysis, bone marrow transplant, and in vitro hematopoietic assays), and improve the leadership and administrative skills required to become a competitive investigator, including grantsmanship. An extensive program of formal training, course-work, meetings with the advisory committee, and dedicated supervision and mentorship by Dr. Daley will ensure that these goals are achieved.